Characterization Of Increased Mucus Production Of HT29 ... - PLOS

Increased MUC2/MUC5AC ratio in SWMS-cultured HT29-MTX-E12 cells

We investigated the expression of genes encoding for mucins in HT29-MTX-E12 cells, since we know from previous studies that culturing this cell type under SWMS conditions led to increased MUC2 production [27]. Indeed, in our study, SWMS conditions resulted in a significant 1.42-fold upregulation of MUC2 (p < 0.05) (Fig 2A). Expression of gel-forming mucins MUC5AC and MUC5B was decreased in response to SWMS (FC = -1.56 and -1.84, respectively, p < 1 • 10−4) (Fig 2A, S1 File). Transmembrane mucins were also significantly lower expressed after SWMS conditions (Fig 2A). MUC3A, 13, 17, 20 and 21 showed FC values between -1.52 and -2.32 (S1 File). Eight out of 19 mucins (MUC4, 6, 7, 12, 15, 16, 19 and 22) displayed very low expression levels under both static and SWMS conditions (RMA < 4) and were not significantly differentially expressed (S1 File). Taken together, these data indicate that SWMS conditions resulted in a change in expression of mucin encoding genes with an increased expression of MUC2, while other gel-forming as well as transmembrane mucins were significantly lower or not differentially expressed.

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Fig 2. Mucin gene and protein expression in HT29-MTX-E12 cells grown under static and SWMS conditions.

A) Microarray gene expression values (Log2) of a panel of secretory and transmembrane mucins B) Protein expression of MUC2 and MUC5AC, expressed as density (a.u.) per μg/mL protein blotted, after correction of Ponceau Red density. Blot data in S2 Fig) C) Ratio of MUC2 and MUC5AC protein expression. * p < 0.05; *** p < 0.001; **** p < 0.0001; n = 3 biological replicates.

https://doi.org/10.1371/journal.pone.0261191.g002

Dot Blot data supported microarray data for MUC2, as protein levels also showed a significant upregulation (p < 0.05) under SWMS versus static conditions (Fig 2B, S2A and S2C Fig). Less MUC5AC was detected under SWMS conditions, however, variation between replicates was rather high and differences were not significant (p = 0.20) (Fig 2B and S2B and S2C Fig). Overall, HT29-MTX-E12 cells grown under SWMS conditions showed a significantly increased MUC2/MUC5AC-ratio (Fig 2C, p < 0.05), confirming previous findings [27]. For LS174T, which we included as a control cell line reported to produce predominantly MUC2, a relatively high MUC2/MUC5AC ratio was calculated (S2D and S2E Fig).

In contrast, Caco-2 cells, which are not reported to produce mucus, showed low RMA values (< 5) for MUC2 and MUC5AC and protein expression of these mucins was below detection level in both conditions (S2F and S2G Fig). Furthermore, similar to HT29-MTX-E12 cells, SWMS conditions resulted in significant downregulation of MUC20, MUC3A and MUC13 in this cell line (S1 File).